The growth of normal cells in vitro and in vivo is regulated at a specific point in the G1 phase of the cell cycle termed the restriction point (R point). Transit through this R point is modulated by growth-regulatory hormones and factors normally supplied by the serum supplement to the growth medium. Cells that are transformed by a variety of mechanisms, such as viruses or chemicals, or that are isolated from tumors in vivo showed a reduced requirement for serum and/or growth factors and are hypothesized to be deficient in their R point regulation. The goal of the project is the determination of the specific hormone and growth factor requirements for R point transit in several phenotypically normal cell lines and the elucidation of their mechanisms of action. We will then establish the relationships between the loss of growth factor requirements and the acquisition, after mutagenesis and selection in vitro, of a transformed, growth-regulated phenotype. We have isolated the phenotypically normal cell lines from embryos of strains of inbred mice. They have been characterized, and defined serum-free media for each are being developed. Mutants that are independent of insulin and epidermal growth factor (EGF) are currently being selected and will be tested for malignant phenotype. In the coming year we will continue to define the roles of individual growth factors and select further growth factor-independent and malignant mutants. In addition, we will be screening the growth factor-independent mutants for production of endogenous growth factors and the presence of growth factor receptors. We also will begin a study of cycloheximide-sensitive growth regulatory proteins in both the parental growth factor-requiring and mutant growth factor-independent cell lines.